The process of cell growth and division is divided into four stages that make up the cell cycle: G1, S (DNA synthesis), G2 and M (mitosis). Progression through the cell cycle is a tightly regulated process, and critical to the cell cycle progression are cyclin-dependent kinases (CDKs). CDKs are the catalytic subunits of a large family of serine/threonine protein kinases. Activation of specific CDKs is required for the appropriate progression through a given stage of the cell cycle and into the next stage in the cell cycle. Hence, regulation of CDK activity is pivotal for the correct timing of cell cycle progression and CDK activity is tightly regulated at many levels, including complex formation with cyclins and CDK inhibitors (CDKI), in particular CIP/KIP and INK-type CDKIs, as well as phosphorylation and dephosphorylation. Central to the activation of a given CDK is the requirement for association with cyclins and phosphorylation at a threonine residue in the activation loop (T-loop). Cyclins are synthesized and degraded during the cell cycle (hence their name), so that activation of a particular CDK occurs only when its cyclin partner(s) becomes available. Additionally, many CDKs require phosphorylation of a threonine residue in the activation loop (T-loop) for their activation. In the case of CDK1, CDK2, CDK4 and CDK6 T-loop phosphorylation is mediated by the CDK activating kinase (CAK).
Deregulation of CDK activity forms an important part of many disease states, generally through elevated and/or inappropriate activation, as CDKs are infrequently mutated. Important mechanisms of CDK deregulation include cyclin overexpression. For example, the cyclin D1 gene is frequently amplified in cancer (Fu et al. Endocrinology 145: 5439-5447 (2004)). CDKI expression is frequently lost, for example, through mutational or epigenetic alterations in genes encoding INK4, CIP or KIP CDKIs in cancer (Malumbres and Barbacid, Nature Reviews Cancer 1, 223-231 (2001)).
CDKs are important targets for the design of drugs with antimimotic, antineurodegenerative, antiviral and antitumor effects. A few specific and high-affinity inhibitors of some CDKs have been developed using CDK2 as a model system. One of these is flavopiridol (clinical phase I/II), which has modest selectivity for CDKs over other kinases and inhibits many members of the CDK family (M. D. Losiewicz et al., Biochem. Biophys. Res. Commun., 201, 589-595 (1994)). One compound class that has yielded many CDK-selective ATP antagonists is 2,6,9-trisubstituted purines. Within this group, roscovitine shows good biological and pharmacological properties (clinical phase I/II) (S. Wang et al., Tetrahedron: Asymmetry, 12, 2891-2894 (2001); M. Mapelli et al., J. Med. Chem., 48, 671-679 (2005)). Recently, another class of compounds having a pyrazolo[1,5-a]pyrimidine skeleton has been developed. These compounds show a high potency for inhibiting CDK2, and in some cases were shown to inhibit the growth of human colon tumor cells (D. S. Williamson et al., Bioorg. Med. Chem. Lett., 15, 863-867 (2005)). However, most CDK inhibitors that have been described do not specifically inhibit one CDK. For example, most CDK2 inhibitors also inhibit CDK1, CDKS, as well as CDK7 and CDK9 (P. M. Fischer, Cell Cycle 3: 742-746). It has also been noted, however, that some inhibitors of structurally similar kinases CDK1, CDK2 and CDKS do not inhibit CDK4 and CDK6 (M. Knockaert et al., Trends Pharmacol. Sci., 23, 417-425 (2002)).
CDK7
While CDK1, CDK2, CDK4 and CDK6 are primarily involved in cell-division control, other cyclin-dependent kinases, such as CDK8 and CDK9, largely regulate transcription. CDK7 is unusual in that it is important in transcription, but also acts as the CDK-activating kinase (CAK) (Lolli and Johnson Cell Cycle 4: 572-577 (2005)). The CDK7 CAK complex comprises cyclin H and the ring finger protein MAT1 and is unusual in that its phosphorylation in the T-loop is not required for its activity (R. P. Fisher et al. Cell 83: 47-57 (1995); Devault et al. EMBO J. 14: 5027-5036 (1995)). In transcription regulation, CDK7/Cyclin H/MAT1 are components of the general transcription factor TFIIH, which is required for initiation of transcription of RNA polymerase II-directed genes. As part of the TFIIH complex, CDK7 phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (R. P. Fisher J. Cell Sci. 118: 5171-5180 (2005)). Additionally, TFIIH plays a role in RNA polymerase I-mediated transcription (Iben et al. Cell 109: 297-306 (2002)). Further, CAK or TFIIH-associated CAK phosphorylate several transcription factors to regulate their activities (see, e.g., Chen et al Mol Cell 6: 127-137 (2000); Bour et al PNAS 102: 16608-16613 (2005)). With respect to cell cycle regulation, the CDK7 CAK complex phosphorylates the cell cycle CDKs in the activation segment (T-loop), required for the activation of CDKs involved in cell cycle regulation (Lolli and Johnson Cell Cycle 4: 572-577 (2005)).